- Email: [email protected]
Association study of single nucleotide polymorphisms of MAFB with non-syndromic cleft lip with or without cleft palate in a population in Heilongjiang Province, northern China
Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
Association study of single nucleotide polymorphisms of MAFB with non-syndromic cleft lip with or without cleft palate in a population in Heilongjiang Province, northern China Na Mi a , Yanru Hao a , Xiaohui Jiao a,∗ , Xudong Zheng b , Tao Song a , Jinna Shi c , Chen Dong a a b c
The Department of Oral Maxillofacial Surgery, College of Stomatology, Harbin Medical University, Harbin, China The medical department, The Second Affiliated Hospital of Harbin Medical University, Harbin, China The Department of Periodontology, The First Affiliated Hospital, Harbin Medical University, Harbin, China
Accepted 3 June 2014 Available online 25 June 2014
Abstract Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common complex birth defect. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a new gene that may be involved in susceptibility to cleft lip with or without cleft palate (CL/P). To further assess its role in NSCLP, we investigated 3 identified single nucleotide polymorphisms in MAFB (rs13041247, rs6065259, and rs11696257) and examined them for association with NSCLP in 344 patients and 324 healthy controls in a northern Chinese Han population with a high incidence of the syndrome. Peripheral blood samples were taken when patients enrolled in the study and DNA samples were extracted from the blood. The 3 single nucleotide polymorphisms were genotyped using a mini-sequencing method (Snapshot® Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK). We found that rs6065259 was the most important single nucleotide polymorphism in MAFB (OR6065259-AA = 0.45; 95% CI: 0.28 to 0.71; p = 0.0027), followed by rs13041247; however, no association was found between rs11696257 and NSCLP. Our study provides further evidence regarding the role of MAFB variations in the development of NSCLP in this northern Chinese Han population. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: NSCLP; susceptibility gene; MAFB; polymorphisms
Introduction
∗ Corresponding author. The Department of Oral Maxillofacial Surgery, College of Stomatology, Harbin Medical University, No.23, YouZheng Road, NanGang District, Harbin150001, Heilongjiang Province, China. Tel.: +86 451 85553926, +86 18645022828; fax: +86 451 53625108. E-mail addresses: [email protected] (N. Mi), [email protected] (Y. Hao), jiaoxiaohui [email protected], jiaoxiaohuidoctor [email protected] (X. Jiao), [email protected] (X. Zheng), [email protected] (T. Song), [email protected] (J. Shi), [email protected] (C. Dong).
Cleft lip with or without cleft palate (CL/P) is one of the most common human birth defects, and the mean worldwide incidence ranges from 1 – 7/1000 live births. In addition, its incidence varies widely across geographical regions and ethnic groups.1 In general, the incidence is highest in Native American and Asian populations (sometimes reaching one or more/500 live births) followed by white populations; those that originate in Africa have the lowest incidence, around 1/2500 births.2 Prevalence is high among Chinese newborns (1.42/1000).3
http://dx.doi.org/10.1016/j.bjoms.2014.06.003 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
747
Table 1 Variables of patients and controls. Hospital
CPO CLP+CLO Mean (SD) age Male Female
1
2
3
66 105 6.3 (6.4) 107 64
32 65 4.1 (4.9) 64 33
24 52 3.2 (3.1) 45 31
All patients (n = 344)
Control (n = 324)
p value
122 222 5.0 (5.1) 216 128
5.2 (5.1) 198 126
– 0.60a 0.66b
Hospital 1: Affiliated Stomatology Hospital of Harbin Medical University. Hospital 2: Second Affiliated Hospital of Harbin Medical University. Hospital 3: Harbin Children’s Hospital. a Independent sample t test. b Two-sided chi square test.
Although CL/P can occur in many Mendelian malformation syndromes, isolated NSCLP, which forms without the involvement of other syndromes, constitutes 70% of all cases.4 Recently, a genome-wide association study found that a new gene, MAFB, may be involved in susceptibility to the syndrome. Beaty et al showed that single nucleotide polymorphisms in or near MAFB have an association with NSCLP, and an analysis of families with Asian ancestry from multiple populations provided strong evidence for the involvement of the gene.5 Several similar studies have been done in the United States, Brazil, Colombia, and central China,6–9 but the results have been inconsistent. We evaluated the 3 single nucleotide polymorphisms (rs13041247, rs6065259, and rs11696257) of MAFB reported by Beaty et al,5 and investigated whether they were associated with NSCLP in this northern Chinese Han population.
Sample This ongoing hospital-based, case-control study involved 344 patients with NSCLP and 324 healthy controls. Patients were recruited from 3 participating sites in the Smile Train Project: The Affiliated Stomatology Hospital of Harbin Medical University, the Second Affiliated Hospital of Harbin Medical University, and Harbin Children’s Hospital, where most patients with orofacial clefts in Heilongjiang Province and the surrounding areas attend clinics. We also randomly selected 324 unrelated healthy controls matched for age, sex, nationality, and residence (urban or rural) from the 3 hospitals after rigorous screening (Table 1). Given that different types of NSCLP are distinct diseases in terms of aetiology and pathogenesis, it is possible that the single nucleotide polymorphisms have unequal effects.10 In our study, the group of patients (n = 344) was conventionally categorised into 2 subgroups: those with CL/P (n = 222) and those with cleft palate only (CPO) (n = 122).
Material and methods
Selection of single nucleotide polymorphisms, DNA extraction, and genotyping
Ethics statement Based on the research of Beaty et al,5 we selected 3 single nucleotide polymorphisms (rs13041247, rs6065259, and rs11696257) of MAFB. Primary information about them is given in Table 2. DNA samples were extracted from peripheral blood using a DNA extraction kit according to the manufacturer’s
The study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University. It complies with the Declaration of Helsinki. We obtained the informed consent of all participants before the study began.
Table 2 Allelic distribution of the single nucleotide polymorphisms (SNP) of the MAFB gene. SNP 6065259 13041247 11696257 a b c
Control MAFa
p valuec
NSCLP MAFa
p valueb
CL/P MAFa
p valueb
CPO MAFa
p valueb
0.4875 0.4906 0.4871
0.74 0.91 0.26
0.3952 0.4298 0.4560
0.001 0.006 0.055
0.4058 0.4372 0.4699
0.012 0.028 0.210
0.3750 0.4158 0.4307
0.006 0.024 0.043
MAF, (minor allele frequency) in the HapMap database for the Han Chinese in Beijing (CHB) population. chi square test. Hardy-Weinberg equilibrium.
748
N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
protocol (Axygen Biosciences, Union City, USA). The DNA pellet was dissolved in Tris-EDTA buffer, and the purity and concentration were identified by spectrophotometric measurement of the absorbance at 260 nm and 280 nm. Genomic DNA was diluted to working concentrations of 50 ng/ml for the genotyping assays. The 3 single nucleotide polymorphisms were genotyped using the mini-sequencing method (Snapshot® Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK) and were analysed using an ABI PRISM 3730 DNA Sequencer according to the manufacturer’s instructions (Applied Biosystems, Foster City, USA). For rs13041247 (T > C), a 303-bp fragment of rs13041247 was amplified from genomic DNA using the primers 5’-AGACCCAGGCCAGGTAGATT-3’ (forward) and 5’CAGGACTTAGCCACCAGAGC-3’ (reverse). Probes for detection of the polymorphism were TCTTCTTGTACTTCCTGGCTG. For rs6065259 (G > A), a 269-bp fragment of rs6065259 was amplified from genomic DNA using the primers 5’-TGCAGATGTGGATGGTGACT-3’ (forward) and 5’-AGGGGGAGAATCTCTGAGGA-3’ (reverse), and the probes were CCTATTTGGTGCTTATTACCCT. Likewise, for rs11696257 (C > T), a 241-bp fragment of rs13169257 was amplified from genomic DNA using the primers 5’-CTGCAAATCTGCCCCTAAAG -3’ (forward) and 5’-AGGCATCTCTGAGGAGGTGA -3’ (reverse), and the probes were AGGCCTCCAGGCCTTTGC. To validate identification of the genotype, 20% of the samples were randomly repeated and the results were fully consistent.
Fig. 1. Linkage disequilibrium blocks for the MAFB haplotype analysis.
respectively). There were also differences in the allele frequencies of rs11696257 in the CPO subgroup. Distribution of genotype frequencies
Statistical analyses The chi square test was used to evaluate differences in the minor allele frequencies and genotype of the single nucleotide polymorphisms between patients and controls (SAS software version 9.1.3, SAS Institute Inc, Cary, USA). All allele and genotype frequency, Hardy–Weinberg equilibrium, pairwise linkage disequilibrium, and haplotype analyses were done online using a web-based association study program.11 The association studies were based on logistic regression analyses in accordance with the individual single nucleotide polymorphisms (response variables), and included analyses of individual and multiple single nucleotide polymorphisms.12 Probabilities of less than 0.05 (two-sided) were considered significant.
Results Distribution of allele frequencies All the single nucleotide polymorphisms were in HardyWeinberg equilibrium. The single nucleotide polymorphism information and allele frequencies are listed in Table 2. There were significant differences between the patients and controls in the allele frequencies of rs6065259 and rs13041247 (1 degree of freedom; p = 0.0012 and p = 0.0059,
To estimate the relative risk of developing NSCLP, odds ratios (OR) were calculated to compare the genotype frequencies of the patients and controls and, according to the Akaike information criterion,13 to find the loci of the most appropriate genetic model associated with risk of the condition (Table 3). The results of the statistical analyses indicate that there were significant differences in the frequencies of the AA genotype of rs6065259 and the CC genotype of rs13041247 (OR6065259-AA = 0.45, 95% CI: 0.28 to 0.71; OR6065259-AA+AG = 0.65, 95% CI: 0.47 to 0.92, p = 0.0027, and OR13041247-CC = 0.50, 95% CI: 0.32 to 0.79, p = 0.0098), and these genotypes were associated with a decreased risk of NSCLP. When the patients were divided into the subgroups, there was a similar significant trend between the patients and controls for rs6065259 in the CL/P subgroup (ORAA = 0.54, 95% CI: 0.33 to 0.89, p = 0.033). In the CPO subgroup there were significant differences in the genotypes of the 3 single nucleotide polymorphisms (1 degree of freedom, p6065259 = 0.0005, p13041247 = 0.010, and p11696257 = 0.023). The gene haplotypes and NSCLP There was strong pairwise linkage disequilibrium between the 3 single nucleotide polymorphisms (0.80 < R2 < 1) and they were in one block (Fig. 1). When R2 is closer to one, the linkage disequilibrium is stronger. An analysis based on
N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
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Table 3 Adjusted OR (95% CI) for association between the single nucleotide polymorphisms (SNP) in MAFB NSCLP. SNP
Genotype
6065259
13041247
11696257
G/G G/A A/A G/A-A/A T/T T/C C/C T/C-C/C C/C C/T T/T C/T-T/T
NSCLP
CL/P
CPO
OR (95% CI)
p value
OR (95% CI)
p value
OR (95% CI)
p value
1.00 (Ref) 0.75 (0.52 to 1.06) 0.45 (0.28 to 0.71) 0.65 (0.47 to 0.92) 1.00 (Ref) 0.78 (0.54 to 1.13) 0.50 (0.32 to 0.79) 0.69 (0.49 to 0.97) 1.00 (Ref) 0.82 (0.56 to 1.22) 0.60 (0.37 to 0.98) 0.76 (0.52 to 1.10)
0.0027
1.00 (Ref) 0.67 (0.45 to 0.99) 0.54 (0.33 to 0.89) 0.63 (0.43 to 0.91) 1.00 (Ref) 0.74 (0.49 to 1.11) 0.56 (0.34 to 0.93) 0.76 (0.51 to 1.15) 1.00 (Ref) 0.78 (0.51 to 1.19) 0.74 (0.44 to 1.22) 0.76 (0.51 to 1.15)
0.033
1.00 (Ref) 0.91 (0.56 to 1.46) 0.26 (0.12 to 0.58) 0.71 (0.45 to 1.12) 1.00 (Ref) 0.86 (0.53 to 1.40) 0.39 (0.20 to 0.76) 0.70 (0.44 to 1.12) 1.00 (Ref) 0.94 (0.56 to 1.56) 0.43 (0.22 to 0.87) 0.78 (0.48 to 1.27)
0.0005
0.0098
0.14
haplotypes can be advantageous over that based on individual single nucleotide polymorphisms14 so we analysed haplotypes for rs6065259-rs13041247-rs11696257 (Table 4). In the NSCLP and CPO groups, there were differences in the haplotype frequencies between the patients and controls (global p values of 0.0002, and <0.0001, respectively). For the A-C-T haplotype, there were significant differences between the 3 groups; its frequency in the total patient set was lower than that of the total control set. The OR was 0.71 (95% CI: 0.55 to 0.90). In the CPO group, differences in the G-T-T haplotype frequencies were significant compared with the control group; the frequency of this haplotype in the CPO group (6.4%) was more than that in the total control set (1.0%).
Discussion NSCLP is a complex, polygenetic, hereditary disease. To date, about 20% of the factors for genetic susceptibility have been identified,5 but the identification of susceptibility genes from different studies has been inconsistent. A large amount of progress has been made in the identification of putative candidate genes for NSCLP, and recently, several genome-wide association studies have identified new loci associated with CL/P (on 8q24, 17q22, and 10q25.3), as well as those near the MAFB and ABCA4 genes.5,15–17 We aimed
0.071
0.42
0.01
0.023
to investigate whether human MAFB polymorphisms were associated with NSCLP in this northern Chinese Han population, and to the best of our knowledge, we have shown the first evidence that loci near MAFB (rs6065259 and rs13041247) reduced the risk. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B; 20q11.2-q13.1) is a transcription factor that has been shown to have an important role in the development of hindbrain structures, the thymus, interneurons, pancreatic islet cells, and the haematopoietic system.18 It encodes a basic leucine zipper (bZIP) transcription factor that has an important role in the regulation of haematopoietic lineage-specific genes, and also functions as an oncogene responsible for the transformation of MAF-expressing myeloma cells.19,20 Recently, studies to identify it were done in mice. MAFB mRNA and protein are expressed in the craniofacial ectoderm and the mesoderm derived from the neural crest between embryonic day 13.5 and 14.5,5 which suggests that the gene may have a role in NSCLP. Compared with the study by Beaty et al, we did not find any associations between rs11696257 and susceptibility to NSCLP; the minor allele frequency for this single nucleotide polymorphism among our controls was 0.4871, which is different from that of 0.414 reported in an Asian population.5 In addition, the minor allele frequencies for rs13041247 and rs6065259 were different in the patients and
Table 4 Association between the common MAFB haplotypes (rs6065259-rs13041247-rs11696257) and NSCLP. Haplotype
GTC ACT GCT GTT ACC ATC Rareb a b
NSCLP
CL/P
OR (95% CI)
p value
1.00 (Ref) 0.71 (0.55 to 0.90) 1.30 (0.73 to 2.30) 3.53 (1.42 to 8.77) 1.58 (0.56 to 4.46) 1.09 (0.35 to 3.35) 1.62 (0.56 to 4.69)
– 0.005 0.37 0.007 0.38 0.89 0.37
a
CPO
OR (95% CI)
p value
1.00 (Ref) 0.76 (0.58 to 0.99) 1.39 (0.75 to 2.59) 2.63 (0.99 to 6.97) – 1.26 (0.38 to 4.22) 1.12 (0.44 to 2.83)
– 0.044 0.29 0.052 – 0.71 0.81
Global haplotype association with NSCLP: p = 0.0002; with CL/P: p = 0.018; with CPO p < 0.0001. “Rare” consists of haplotypes with a frequency < 0.01.
a
OR (95% CI)
p valuea
1.00 (Ref) 0.61 (0.43 to 0.88) 1.20 (0.53 to 2.70) 5.32 (1.92 to 14.73) 3.06 (0.95 to 9.86) – 1.13 (0.36 to 3.53)
– 0.008 0.67 0.001 0.062 – 0.83
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N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
controls (0.4906 compared with 0.414, and 0.4875 compared with 0.414, respectively). We found that rs6065259, which is located near MAFB, was the single nucleotide polymorphism most significantly associated with NSCLP, followed by rs13041247. These findings are notably different from those of Beaty et al.5 Our findings also suggest that these single nucleotide polymorphisms have different roles in the development of NSCLP in the northern Chinese Han population. When we further stratified the patients into 2 subgroups (CL/P and CPO) we found that only rs6065259 was associated with both subgroups, and the minor allele exhibited a protective effect. Interestingly, all 3 single nucleotide polymorphisms were associated with the CPO subgroup, and the minor allele exhibited a protective effect. Therefore, it is possible that MAFB may have an important role in the development of NSCLP, and particularly in the development of CPO, in the population studied. Analyses of haplotypes further confirmed the role of MAFB in the development of NSCLP. The haplotype A-C-T exhibited a protective effect on the risk of developing NSCLP, and interestingly, patients with the haplotype G-T-T had an increased risk of developing CPO. In conclusion, despite the fact that our study was limited by the moderate sample size, our findings confirm that the MAFB polymorphisms were significantly associated with the risk of NSCLP in the population studied, which is consistent with previous genome-wide association studies, and our results improve our understanding of NSCLP. MAFB remains an interesting candidate gene and it clearly warrants further study. In the future, a well-designed study that investigates the interaction of the MAFB gene and the environment with different ethnic groups could verify its role in NSCLP.
Conflict of Interest We have no conflicts of interest.
Ethics statement The study was reviewed and approved by the Ethics Committee of The Medical University. It complies with the Declaration of Helsinki. Informed consent was obtained from all participants before the study began.
Ethics statement/confirmation of patient permission The study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University. The study complies with the Declaration of Helsinki. Informed consent was obtained from subjects before the initiation of the study.
Acknowledgements This research was supported by the Natural Science Foundation of Heilongjiang Province (ZD201212).
References 1. Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374:1773–85. 2. Stanier P, Moore GE. Genetics of cleft lip and palate: syndromic genes contribute to the incidence of non-syndromic clefts. Hum Mol Genet 2004;13:R73–81. 3. Dai L, Zhu J, Mao M, et al. Time trends in oral clefts in Chinese newborns: data from the Chinese National Birth Defects Monitoring Network. Birth Defects Res A Clin Mol Teratol 2010;88:41–7. 4. Sivertsen A, Wilcox AJ, Skjærven R, et al. Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. BMJ 2008;336:432–4. 5. Beaty TH, Murray JC, Marazita ML, et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet 2010;42:525–9. 6. Lennon CJ, Birkeland AC, Nu˜nez JA, et al. Association of candidate genes with nonsyndromic clefts in Honduran and Colombian populations. Laryngoscope 2012;122:2082–7. 7. Fontoura C, Silva RM, Granjeiro JM, Letra A. Further evidence of association of the ABCA4 gene with cleft lip/palate. Eur J Oral Sci 2012;120:553–7. 8. Pan Y, Zhang W, Du Y, et al. Different roles of two novel susceptibility loci for nonsyndromic orofacial clefts in a Chinese Han population. Am J Med Genet A 2011;155A:2180–5. 9. Yuan Q, Blanton SH, Hecht JT. Association of ABCA4 and MAFB with non-syndromic cleft lip with or without cleft palate. Am J Med Genet A 2011;155A:1469–71. 10. Rahimov F, Marazita ML, Visel A, et al. Disruption of an AP-2alpha binding site in an IRF6 enhancer is associated with cleft lip. Nat Genet 2008;40:1341–7. 11. SNPStats. Institut Català d’Oncologia. Available from URL: http:// bioinfo.iconcologia.net/snpstats/start.htm 12. Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics 2006;22:1928–9. 13. Akaike H. A new look at the statistical model identification. IEEE Transactions on Automatic Control 1974;19:716–23. 14. Morris RW, Kaplan NL. On the advantage of haplotype analysis in the presence of multiple disease susceptibility alleles. Genet Epidemiol 2002;23:221–33. 15. Birnbaum S, Ludwig KU, Reutter H, et al. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet 2009;41:473–7. 16. Grant SF, Wang K, Zhang H, et al. A genome-wide association study identifies a locus for nonsyndromic cleft lip with or without cleft palate on 8q24. J Pediatr 2009;155:909–13. 17. Mangold E, Ludwig KU, Birnbaum S, et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet 2010;42:24–6. 18. Yang Y, Cvekl A. Large Maf transcription factors: cousins of AP-1 proteins and important regulators of cellular differentiation. Einstein J Biol Med 2007;23:2–11. 19. Gemelli C, Montanari M, Tenedini E, et al. Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells. Cell Death Differ 2006;13:1686–96. 20. van Stralen E, van de Wetering M, Agnelli L, Neri A, Clevers HC, Bast BJ. Identification of primary MAFB target genes in multiple myeloma. Exp Hematol 2009;37:78–86.
British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
Association study of single nucleotide polymorphisms of MAFB with non-syndromic cleft lip with or without cleft palate in a population in Heilongjiang Province, northern China Na Mi a , Yanru Hao a , Xiaohui Jiao a,∗ , Xudong Zheng b , Tao Song a , Jinna Shi c , Chen Dong a a b c
The Department of Oral Maxillofacial Surgery, College of Stomatology, Harbin Medical University, Harbin, China The medical department, The Second Affiliated Hospital of Harbin Medical University, Harbin, China The Department of Periodontology, The First Affiliated Hospital, Harbin Medical University, Harbin, China
Accepted 3 June 2014 Available online 25 June 2014
Abstract Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common complex birth defect. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a new gene that may be involved in susceptibility to cleft lip with or without cleft palate (CL/P). To further assess its role in NSCLP, we investigated 3 identified single nucleotide polymorphisms in MAFB (rs13041247, rs6065259, and rs11696257) and examined them for association with NSCLP in 344 patients and 324 healthy controls in a northern Chinese Han population with a high incidence of the syndrome. Peripheral blood samples were taken when patients enrolled in the study and DNA samples were extracted from the blood. The 3 single nucleotide polymorphisms were genotyped using a mini-sequencing method (Snapshot® Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK). We found that rs6065259 was the most important single nucleotide polymorphism in MAFB (OR6065259-AA = 0.45; 95% CI: 0.28 to 0.71; p = 0.0027), followed by rs13041247; however, no association was found between rs11696257 and NSCLP. Our study provides further evidence regarding the role of MAFB variations in the development of NSCLP in this northern Chinese Han population. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: NSCLP; susceptibility gene; MAFB; polymorphisms
Introduction
∗ Corresponding author. The Department of Oral Maxillofacial Surgery, College of Stomatology, Harbin Medical University, No.23, YouZheng Road, NanGang District, Harbin150001, Heilongjiang Province, China. Tel.: +86 451 85553926, +86 18645022828; fax: +86 451 53625108. E-mail addresses: [email protected] (N. Mi), [email protected] (Y. Hao), jiaoxiaohui [email protected], jiaoxiaohuidoctor [email protected] (X. Jiao), [email protected] (X. Zheng), [email protected] (T. Song), [email protected] (J. Shi), [email protected] (C. Dong).
Cleft lip with or without cleft palate (CL/P) is one of the most common human birth defects, and the mean worldwide incidence ranges from 1 – 7/1000 live births. In addition, its incidence varies widely across geographical regions and ethnic groups.1 In general, the incidence is highest in Native American and Asian populations (sometimes reaching one or more/500 live births) followed by white populations; those that originate in Africa have the lowest incidence, around 1/2500 births.2 Prevalence is high among Chinese newborns (1.42/1000).3
http://dx.doi.org/10.1016/j.bjoms.2014.06.003 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
747
Table 1 Variables of patients and controls. Hospital
CPO CLP+CLO Mean (SD) age Male Female
1
2
3
66 105 6.3 (6.4) 107 64
32 65 4.1 (4.9) 64 33
24 52 3.2 (3.1) 45 31
All patients (n = 344)
Control (n = 324)
p value
122 222 5.0 (5.1) 216 128
5.2 (5.1) 198 126
– 0.60a 0.66b
Hospital 1: Affiliated Stomatology Hospital of Harbin Medical University. Hospital 2: Second Affiliated Hospital of Harbin Medical University. Hospital 3: Harbin Children’s Hospital. a Independent sample t test. b Two-sided chi square test.
Although CL/P can occur in many Mendelian malformation syndromes, isolated NSCLP, which forms without the involvement of other syndromes, constitutes 70% of all cases.4 Recently, a genome-wide association study found that a new gene, MAFB, may be involved in susceptibility to the syndrome. Beaty et al showed that single nucleotide polymorphisms in or near MAFB have an association with NSCLP, and an analysis of families with Asian ancestry from multiple populations provided strong evidence for the involvement of the gene.5 Several similar studies have been done in the United States, Brazil, Colombia, and central China,6–9 but the results have been inconsistent. We evaluated the 3 single nucleotide polymorphisms (rs13041247, rs6065259, and rs11696257) of MAFB reported by Beaty et al,5 and investigated whether they were associated with NSCLP in this northern Chinese Han population.
Sample This ongoing hospital-based, case-control study involved 344 patients with NSCLP and 324 healthy controls. Patients were recruited from 3 participating sites in the Smile Train Project: The Affiliated Stomatology Hospital of Harbin Medical University, the Second Affiliated Hospital of Harbin Medical University, and Harbin Children’s Hospital, where most patients with orofacial clefts in Heilongjiang Province and the surrounding areas attend clinics. We also randomly selected 324 unrelated healthy controls matched for age, sex, nationality, and residence (urban or rural) from the 3 hospitals after rigorous screening (Table 1). Given that different types of NSCLP are distinct diseases in terms of aetiology and pathogenesis, it is possible that the single nucleotide polymorphisms have unequal effects.10 In our study, the group of patients (n = 344) was conventionally categorised into 2 subgroups: those with CL/P (n = 222) and those with cleft palate only (CPO) (n = 122).
Material and methods
Selection of single nucleotide polymorphisms, DNA extraction, and genotyping
Ethics statement Based on the research of Beaty et al,5 we selected 3 single nucleotide polymorphisms (rs13041247, rs6065259, and rs11696257) of MAFB. Primary information about them is given in Table 2. DNA samples were extracted from peripheral blood using a DNA extraction kit according to the manufacturer’s
The study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University. It complies with the Declaration of Helsinki. We obtained the informed consent of all participants before the study began.
Table 2 Allelic distribution of the single nucleotide polymorphisms (SNP) of the MAFB gene. SNP 6065259 13041247 11696257 a b c
Control MAFa
p valuec
NSCLP MAFa
p valueb
CL/P MAFa
p valueb
CPO MAFa
p valueb
0.4875 0.4906 0.4871
0.74 0.91 0.26
0.3952 0.4298 0.4560
0.001 0.006 0.055
0.4058 0.4372 0.4699
0.012 0.028 0.210
0.3750 0.4158 0.4307
0.006 0.024 0.043
MAF, (minor allele frequency) in the HapMap database for the Han Chinese in Beijing (CHB) population. chi square test. Hardy-Weinberg equilibrium.
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protocol (Axygen Biosciences, Union City, USA). The DNA pellet was dissolved in Tris-EDTA buffer, and the purity and concentration were identified by spectrophotometric measurement of the absorbance at 260 nm and 280 nm. Genomic DNA was diluted to working concentrations of 50 ng/ml for the genotyping assays. The 3 single nucleotide polymorphisms were genotyped using the mini-sequencing method (Snapshot® Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK) and were analysed using an ABI PRISM 3730 DNA Sequencer according to the manufacturer’s instructions (Applied Biosystems, Foster City, USA). For rs13041247 (T > C), a 303-bp fragment of rs13041247 was amplified from genomic DNA using the primers 5’-AGACCCAGGCCAGGTAGATT-3’ (forward) and 5’CAGGACTTAGCCACCAGAGC-3’ (reverse). Probes for detection of the polymorphism were TCTTCTTGTACTTCCTGGCTG. For rs6065259 (G > A), a 269-bp fragment of rs6065259 was amplified from genomic DNA using the primers 5’-TGCAGATGTGGATGGTGACT-3’ (forward) and 5’-AGGGGGAGAATCTCTGAGGA-3’ (reverse), and the probes were CCTATTTGGTGCTTATTACCCT. Likewise, for rs11696257 (C > T), a 241-bp fragment of rs13169257 was amplified from genomic DNA using the primers 5’-CTGCAAATCTGCCCCTAAAG -3’ (forward) and 5’-AGGCATCTCTGAGGAGGTGA -3’ (reverse), and the probes were AGGCCTCCAGGCCTTTGC. To validate identification of the genotype, 20% of the samples were randomly repeated and the results were fully consistent.
Fig. 1. Linkage disequilibrium blocks for the MAFB haplotype analysis.
respectively). There were also differences in the allele frequencies of rs11696257 in the CPO subgroup. Distribution of genotype frequencies
Statistical analyses The chi square test was used to evaluate differences in the minor allele frequencies and genotype of the single nucleotide polymorphisms between patients and controls (SAS software version 9.1.3, SAS Institute Inc, Cary, USA). All allele and genotype frequency, Hardy–Weinberg equilibrium, pairwise linkage disequilibrium, and haplotype analyses were done online using a web-based association study program.11 The association studies were based on logistic regression analyses in accordance with the individual single nucleotide polymorphisms (response variables), and included analyses of individual and multiple single nucleotide polymorphisms.12 Probabilities of less than 0.05 (two-sided) were considered significant.
Results Distribution of allele frequencies All the single nucleotide polymorphisms were in HardyWeinberg equilibrium. The single nucleotide polymorphism information and allele frequencies are listed in Table 2. There were significant differences between the patients and controls in the allele frequencies of rs6065259 and rs13041247 (1 degree of freedom; p = 0.0012 and p = 0.0059,
To estimate the relative risk of developing NSCLP, odds ratios (OR) were calculated to compare the genotype frequencies of the patients and controls and, according to the Akaike information criterion,13 to find the loci of the most appropriate genetic model associated with risk of the condition (Table 3). The results of the statistical analyses indicate that there were significant differences in the frequencies of the AA genotype of rs6065259 and the CC genotype of rs13041247 (OR6065259-AA = 0.45, 95% CI: 0.28 to 0.71; OR6065259-AA+AG = 0.65, 95% CI: 0.47 to 0.92, p = 0.0027, and OR13041247-CC = 0.50, 95% CI: 0.32 to 0.79, p = 0.0098), and these genotypes were associated with a decreased risk of NSCLP. When the patients were divided into the subgroups, there was a similar significant trend between the patients and controls for rs6065259 in the CL/P subgroup (ORAA = 0.54, 95% CI: 0.33 to 0.89, p = 0.033). In the CPO subgroup there were significant differences in the genotypes of the 3 single nucleotide polymorphisms (1 degree of freedom, p6065259 = 0.0005, p13041247 = 0.010, and p11696257 = 0.023). The gene haplotypes and NSCLP There was strong pairwise linkage disequilibrium between the 3 single nucleotide polymorphisms (0.80 < R2 < 1) and they were in one block (Fig. 1). When R2 is closer to one, the linkage disequilibrium is stronger. An analysis based on
N. Mi et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 746–750
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Table 3 Adjusted OR (95% CI) for association between the single nucleotide polymorphisms (SNP) in MAFB NSCLP. SNP
Genotype
6065259
13041247
11696257
G/G G/A A/A G/A-A/A T/T T/C C/C T/C-C/C C/C C/T T/T C/T-T/T
NSCLP
CL/P
CPO
OR (95% CI)
p value
OR (95% CI)
p value
OR (95% CI)
p value
1.00 (Ref) 0.75 (0.52 to 1.06) 0.45 (0.28 to 0.71) 0.65 (0.47 to 0.92) 1.00 (Ref) 0.78 (0.54 to 1.13) 0.50 (0.32 to 0.79) 0.69 (0.49 to 0.97) 1.00 (Ref) 0.82 (0.56 to 1.22) 0.60 (0.37 to 0.98) 0.76 (0.52 to 1.10)
0.0027
1.00 (Ref) 0.67 (0.45 to 0.99) 0.54 (0.33 to 0.89) 0.63 (0.43 to 0.91) 1.00 (Ref) 0.74 (0.49 to 1.11) 0.56 (0.34 to 0.93) 0.76 (0.51 to 1.15) 1.00 (Ref) 0.78 (0.51 to 1.19) 0.74 (0.44 to 1.22) 0.76 (0.51 to 1.15)
0.033
1.00 (Ref) 0.91 (0.56 to 1.46) 0.26 (0.12 to 0.58) 0.71 (0.45 to 1.12) 1.00 (Ref) 0.86 (0.53 to 1.40) 0.39 (0.20 to 0.76) 0.70 (0.44 to 1.12) 1.00 (Ref) 0.94 (0.56 to 1.56) 0.43 (0.22 to 0.87) 0.78 (0.48 to 1.27)
0.0005
0.0098
0.14
haplotypes can be advantageous over that based on individual single nucleotide polymorphisms14 so we analysed haplotypes for rs6065259-rs13041247-rs11696257 (Table 4). In the NSCLP and CPO groups, there were differences in the haplotype frequencies between the patients and controls (global p values of 0.0002, and <0.0001, respectively). For the A-C-T haplotype, there were significant differences between the 3 groups; its frequency in the total patient set was lower than that of the total control set. The OR was 0.71 (95% CI: 0.55 to 0.90). In the CPO group, differences in the G-T-T haplotype frequencies were significant compared with the control group; the frequency of this haplotype in the CPO group (6.4%) was more than that in the total control set (1.0%).
Discussion NSCLP is a complex, polygenetic, hereditary disease. To date, about 20% of the factors for genetic susceptibility have been identified,5 but the identification of susceptibility genes from different studies has been inconsistent. A large amount of progress has been made in the identification of putative candidate genes for NSCLP, and recently, several genome-wide association studies have identified new loci associated with CL/P (on 8q24, 17q22, and 10q25.3), as well as those near the MAFB and ABCA4 genes.5,15–17 We aimed
0.071
0.42
0.01
0.023
to investigate whether human MAFB polymorphisms were associated with NSCLP in this northern Chinese Han population, and to the best of our knowledge, we have shown the first evidence that loci near MAFB (rs6065259 and rs13041247) reduced the risk. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B; 20q11.2-q13.1) is a transcription factor that has been shown to have an important role in the development of hindbrain structures, the thymus, interneurons, pancreatic islet cells, and the haematopoietic system.18 It encodes a basic leucine zipper (bZIP) transcription factor that has an important role in the regulation of haematopoietic lineage-specific genes, and also functions as an oncogene responsible for the transformation of MAF-expressing myeloma cells.19,20 Recently, studies to identify it were done in mice. MAFB mRNA and protein are expressed in the craniofacial ectoderm and the mesoderm derived from the neural crest between embryonic day 13.5 and 14.5,5 which suggests that the gene may have a role in NSCLP. Compared with the study by Beaty et al, we did not find any associations between rs11696257 and susceptibility to NSCLP; the minor allele frequency for this single nucleotide polymorphism among our controls was 0.4871, which is different from that of 0.414 reported in an Asian population.5 In addition, the minor allele frequencies for rs13041247 and rs6065259 were different in the patients and
Table 4 Association between the common MAFB haplotypes (rs6065259-rs13041247-rs11696257) and NSCLP. Haplotype
GTC ACT GCT GTT ACC ATC Rareb a b
NSCLP
CL/P
OR (95% CI)
p value
1.00 (Ref) 0.71 (0.55 to 0.90) 1.30 (0.73 to 2.30) 3.53 (1.42 to 8.77) 1.58 (0.56 to 4.46) 1.09 (0.35 to 3.35) 1.62 (0.56 to 4.69)
– 0.005 0.37 0.007 0.38 0.89 0.37
a
CPO
OR (95% CI)
p value
1.00 (Ref) 0.76 (0.58 to 0.99) 1.39 (0.75 to 2.59) 2.63 (0.99 to 6.97) – 1.26 (0.38 to 4.22) 1.12 (0.44 to 2.83)
– 0.044 0.29 0.052 – 0.71 0.81
Global haplotype association with NSCLP: p = 0.0002; with CL/P: p = 0.018; with CPO p < 0.0001. “Rare” consists of haplotypes with a frequency < 0.01.
a
OR (95% CI)
p valuea
1.00 (Ref) 0.61 (0.43 to 0.88) 1.20 (0.53 to 2.70) 5.32 (1.92 to 14.73) 3.06 (0.95 to 9.86) – 1.13 (0.36 to 3.53)
– 0.008 0.67 0.001 0.062 – 0.83
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controls (0.4906 compared with 0.414, and 0.4875 compared with 0.414, respectively). We found that rs6065259, which is located near MAFB, was the single nucleotide polymorphism most significantly associated with NSCLP, followed by rs13041247. These findings are notably different from those of Beaty et al.5 Our findings also suggest that these single nucleotide polymorphisms have different roles in the development of NSCLP in the northern Chinese Han population. When we further stratified the patients into 2 subgroups (CL/P and CPO) we found that only rs6065259 was associated with both subgroups, and the minor allele exhibited a protective effect. Interestingly, all 3 single nucleotide polymorphisms were associated with the CPO subgroup, and the minor allele exhibited a protective effect. Therefore, it is possible that MAFB may have an important role in the development of NSCLP, and particularly in the development of CPO, in the population studied. Analyses of haplotypes further confirmed the role of MAFB in the development of NSCLP. The haplotype A-C-T exhibited a protective effect on the risk of developing NSCLP, and interestingly, patients with the haplotype G-T-T had an increased risk of developing CPO. In conclusion, despite the fact that our study was limited by the moderate sample size, our findings confirm that the MAFB polymorphisms were significantly associated with the risk of NSCLP in the population studied, which is consistent with previous genome-wide association studies, and our results improve our understanding of NSCLP. MAFB remains an interesting candidate gene and it clearly warrants further study. In the future, a well-designed study that investigates the interaction of the MAFB gene and the environment with different ethnic groups could verify its role in NSCLP.
Conflict of Interest We have no conflicts of interest.
Ethics statement The study was reviewed and approved by the Ethics Committee of The Medical University. It complies with the Declaration of Helsinki. Informed consent was obtained from all participants before the study began.
Ethics statement/confirmation of patient permission The study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University. The study complies with the Declaration of Helsinki. Informed consent was obtained from subjects before the initiation of the study.
Acknowledgements This research was supported by the Natural Science Foundation of Heilongjiang Province (ZD201212).
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